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CRISPR-Driven Genome Engineering for Chorismate- and Anthranilate-Accumulating Corynebacterium Cell Factories

In this study, we aimed to enhance the accumulation of chorismate (CHR) and anthranilate (ANT), key intermediates in the shikimate pathway, by modifying a shikimate over-producing recombinant strain of Corynebacterium glutamicum [19]. To achieve this, we utilized a CRISPR-driven genome engineering a...

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Autores principales: Kim, Hye-Jin, Choi, Si-Sun, Kim, Eung-Soo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Korean Society for Microbiology and Biotechnology 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10619553/
https://www.ncbi.nlm.nih.gov/pubmed/37463859
http://dx.doi.org/10.4014/jmb.2305.05031
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author Kim, Hye-Jin
Choi, Si-Sun
Kim, Eung-Soo
author_facet Kim, Hye-Jin
Choi, Si-Sun
Kim, Eung-Soo
author_sort Kim, Hye-Jin
collection PubMed
description In this study, we aimed to enhance the accumulation of chorismate (CHR) and anthranilate (ANT), key intermediates in the shikimate pathway, by modifying a shikimate over-producing recombinant strain of Corynebacterium glutamicum [19]. To achieve this, we utilized a CRISPR-driven genome engineering approach to compensate for the deletion of shikimate kinase (AroK) as well as ANT synthases (TrpEG) and ANT phosphoribosyltransferase (TrpD). In addition, we inhibited the CHR metabolic pathway to induce CHR accumulation. Further, to optimize the shikimate pathway, we overexpressed feedback inhibition-resistant Escherichia coli AroG and AroH genes, as well as C. glutamicum AroF and AroB genes. We also overexpressed QsuC and substituted shikimate dehydrogenase (AroE). In parallel, we optimized the carbon metabolism pathway by deleting the gntR family transcriptional regulator (IolR) and overexpressing polyphosphate/ATP-dependent glucokinase (PpgK) and glucose kinase (Glk). Moreover, acetate kinase (Ack) and phosphotransacetylase (Pta) were eliminated. Through our CRISPR-driven genome re-design approach, we successfully generated C. glutamicum cell factories capable of producing up to 0.48 g/l and 0.9 g/l of CHR and ANT in 1.3 ml miniature culture systems, respectively. These findings highlight the efficacy of our rational cell factory design strategy in C. glutamicum, which provides a robust platform technology for developing high-producing strains that synthesize valuable aromatic compounds, particularly those derived from the shikimate pathway metabolites.
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spelling pubmed-106195532023-11-02 CRISPR-Driven Genome Engineering for Chorismate- and Anthranilate-Accumulating Corynebacterium Cell Factories Kim, Hye-Jin Choi, Si-Sun Kim, Eung-Soo J Microbiol Biotechnol Research article In this study, we aimed to enhance the accumulation of chorismate (CHR) and anthranilate (ANT), key intermediates in the shikimate pathway, by modifying a shikimate over-producing recombinant strain of Corynebacterium glutamicum [19]. To achieve this, we utilized a CRISPR-driven genome engineering approach to compensate for the deletion of shikimate kinase (AroK) as well as ANT synthases (TrpEG) and ANT phosphoribosyltransferase (TrpD). In addition, we inhibited the CHR metabolic pathway to induce CHR accumulation. Further, to optimize the shikimate pathway, we overexpressed feedback inhibition-resistant Escherichia coli AroG and AroH genes, as well as C. glutamicum AroF and AroB genes. We also overexpressed QsuC and substituted shikimate dehydrogenase (AroE). In parallel, we optimized the carbon metabolism pathway by deleting the gntR family transcriptional regulator (IolR) and overexpressing polyphosphate/ATP-dependent glucokinase (PpgK) and glucose kinase (Glk). Moreover, acetate kinase (Ack) and phosphotransacetylase (Pta) were eliminated. Through our CRISPR-driven genome re-design approach, we successfully generated C. glutamicum cell factories capable of producing up to 0.48 g/l and 0.9 g/l of CHR and ANT in 1.3 ml miniature culture systems, respectively. These findings highlight the efficacy of our rational cell factory design strategy in C. glutamicum, which provides a robust platform technology for developing high-producing strains that synthesize valuable aromatic compounds, particularly those derived from the shikimate pathway metabolites. The Korean Society for Microbiology and Biotechnology 2023-10-28 2023-07-17 /pmc/articles/PMC10619553/ /pubmed/37463859 http://dx.doi.org/10.4014/jmb.2305.05031 Text en Copyright © 2023 by the authors. Licensee KMB https://creativecommons.org/licenses/by/4.0/This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)
spellingShingle Research article
Kim, Hye-Jin
Choi, Si-Sun
Kim, Eung-Soo
CRISPR-Driven Genome Engineering for Chorismate- and Anthranilate-Accumulating Corynebacterium Cell Factories
title CRISPR-Driven Genome Engineering for Chorismate- and Anthranilate-Accumulating Corynebacterium Cell Factories
title_full CRISPR-Driven Genome Engineering for Chorismate- and Anthranilate-Accumulating Corynebacterium Cell Factories
title_fullStr CRISPR-Driven Genome Engineering for Chorismate- and Anthranilate-Accumulating Corynebacterium Cell Factories
title_full_unstemmed CRISPR-Driven Genome Engineering for Chorismate- and Anthranilate-Accumulating Corynebacterium Cell Factories
title_short CRISPR-Driven Genome Engineering for Chorismate- and Anthranilate-Accumulating Corynebacterium Cell Factories
title_sort crispr-driven genome engineering for chorismate- and anthranilate-accumulating corynebacterium cell factories
topic Research article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10619553/
https://www.ncbi.nlm.nih.gov/pubmed/37463859
http://dx.doi.org/10.4014/jmb.2305.05031
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